【作者】 杨亚军；

【导师】 吴益东；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2010， 博士

【摘要】 棉铃虫Helicoverpa armigera Hubner属鳞翅目夜蛾科,是一种重要的农业害虫。曾经在我国棉花上大面积暴发,造成严重的经济损失。20世纪80年代以来化学杀虫剂的广泛应用,使棉铃虫抗药性得到快速发展。棉铃虫对杀虫剂的抗性,特别是对拟除虫菊酯的抗性剧增是导致20世纪90年代初我国北方棉区棉铃虫大爆发的主要因素之一。随着Bt棉在我国的大面积种植,棉铃虫的发生量得到了有效地控制。但Bt棉在整个生长发育期内都表达Bt毒素,且在我国种植已超过10年。棉铃虫种群如此长时间处于Bt毒素筛选压力之下,对Bt毒素产生抗性的风险日益加大。目前已有多个棉铃虫品系经室内筛选对Bt毒素产生了抗性,如果田间棉铃虫对Bt毒素产生抗性将影响Bt棉的种植寿命。对棉铃虫抗性机理的研究将会为田间棉铃虫抗性治理提供重要依据。钙粘蛋白是Bt毒素作用过程中的一个重要受体,钙粘蛋白的变异将会导致害虫对Bt毒素产生抗性,目前至少已有3种害虫钙粘蛋白的变异被证实与其对Bt毒素的抗性有关。棉铃虫GYBT抗性品系钙粘蛋白基因缺失突变导致其氨基酸在429位提前终止,产生截短蛋白,使Bt毒素失去特异性结合受体。本文对棉铃虫室内抗性品系和田间种群Cry1Ac抗性相关的钙粘蛋白基因突变进行鉴定和检测,评价钙粘蛋白在抗性形成及Bt作用机理中的作用。研究结果不仅对于明确Cry1Ac毒杀棉铃虫的分子机理、进而预测棉铃虫潜在的抗性机理具有重要意义,还有利于了解田间棉铃虫的抗性基因的发展状况,为田间棉铃虫的抗性监测提供技术支持,为棉铃虫对Bt棉花的抗性治理提供重要理论依据。一、棉铃虫室内Cry1Ac抗性品系钙粘蛋白基因突变的鉴定和检测对室内棉铃虫Cry1Ac抗、感品系钙粘蛋白基因组DNA进行克隆和测序,结果显示室内敏感品系GY钙粘蛋白(cadherin, Ha_BtR)基因组DNA (GenBank登录号为DQ523166)全长全长16.4 kb,编码区含有34个外显子、33个内含子。棉铃虫抗性品系GYBT(对Cry1Ac的抗性>500倍)钙粘蛋白Ha_BtR r1等位基因(GenBank登录号为DQ523167)共有8.9 kb。与Ha_BtR野生型s相比在第8外显子和第25外显子之间缺失大约8.8 kb。5’剪切位点位于第8外显子末端,3’剪切位点位于第25外显子前的内含子处。融合区域由第8外显子大部分(88 bp)、第25外显子前内含子部分(120 bp)及第25外显子组成。这一缺失产生了提前终止密码子(TAA),使抗性品系中钙粘蛋白不能完整表达,从而导致抗性产生。针对r1等位基因建立了基于DNA的分子检测方法,这将有利于监测田间棉铃虫种群钙粘蛋白基因r1等位基因的频率。二、田间棉铃虫钙粘蛋白基因突变的鉴定与检测通过F1筛选法对2005年我国河南安阳田间棉铃虫种群及2009年河北廊坊田间种群与Cry1Ac抗性相关钙粘蛋白基因突变进行了筛选、鉴定。在采自2005年我国河南安阳田间棉铃虫种群中我们成功发现了与Cry1Ac抗性相关的钙粘蛋白新的等位基因r2和r3.r2和r3分别在Ha-BtR的第8外显子的相同位置插入了逆转座子(HaRT1)长末端重复(LTR)和完整的逆转座子HaRT1。这导致了钙粘蛋白截短,缺失跨膜区域和毒素结合区。这是首次在田间棉铃虫种群中发现与Cry1Ac抗性相关的钙粘蛋白基因突变,表明钙粘蛋白基因变异可能是田间棉铃虫抗性产生的重要途径。本研究还表明,含这两个抗性等位基因的棉铃虫幼虫在Bt棉上能完成发育过程。钙粘蛋白基因位点应成为田间棉铃虫抗性检测的重要靶标基因之一。同样,通过F1筛选法对2009年河北廊坊地区棉铃虫对Cry1Ac的抗性进行筛查,选出候选单对10对,可疑候选单对4个。对候选单对系F1代抗性个体中来自田间亲本的钙粘蛋白基因全长cDNA进行克隆、测序,发现了5个新的钙粘蛋白基因突变类型。与敏感品系钙粘蛋白基因相比：117#单对系田间亲本钙粘蛋白基因发生插入突变导致在氨基酸754-755间插入30个氨基酸或在氨基酸751处终止；239#在碱基4393-5097位缺失705 bp,导致EC11-CYT处235个氨基酸的缺失；234#发生可变剪接,在碱基1194-2752之间缺失1530 bp,导致EC2-6缺失；256#在碱基2403-1414位缺失导致4个氨基酸缺失(YTIT,802-805位)；124#在碱基4825-4989间缺失165bp,导致在氨基酸1609-1663位间55个氨基酸缺失(CYT部分)。这些突变体的发现表明,与Cry1Ac抗性相关的钙粘蛋白基因突变在田间种群中具有多样性,田间棉铃虫钙粘蛋白不同形式的变异均可能导致抗性的产生,这将增加分子检测钙粘蛋白变异基因频率进而预测抗性发展状况的难度。三、棉铃虫钙粘蛋白近等基因系的构建和交互抗性通过GYBT品系与SCD品系杂交后经多次回交并辅助分子标记筛选将等位基因r1植入SCD品系建立近等基因系SCD-r1.对棉铃虫钙粘蛋白近等基因系SCD与SCD-r1的正反交分析表明SCD-r1对Cry1Ac的抗性是完全隐性、常染色体遗传。抗性品系SCD-r1对Cry1Ac的抗性为高水平抗性(>430倍)；对Cry1Aa、Cry1Ab有中等水平的交互抗性,对Cry2Aa和Cry2Ab2没有交互抗性。原毒素Cry1AcPro和Cry1AbPro分别比胰蛋白酶活化毒素Cry1Ac和Cry1Ab对SCD-r1毒力效果好,SCD-r1品系对原毒素Cry1AcPro和Cry1AbPro的抗性明显低于对胰蛋白酶活化毒素Cry1Ac和Cry1Ab的抗性。修改毒素Cry1AcMod和Cry1AbMod分别比胰蛋白酶活化毒素Cry1Ac和Cry1Ab对SCD-r1毒力效果好,Cry1AcMod和Cry1AbMod都能显著克服棉铃虫对Cry1Ac和Cry1Ab的抗性。这表明钙粘蛋白在与不同毒素及同一毒素不同形式存在不同的相互作用,对Bt毒素进行人工修饰能够克服基于钙粘蛋白基因变异产生的抗性,修饰的Bt毒素基因可能成为转基因抗虫作物的候选基因。四、我国田间棉铃虫对Cry1Ac的敏感性监测用毒素涂表法对我国2006-2008年主要棉区的棉铃虫对Cry1Ac的敏感性进行了监测。2006-2008年监测的12个田间种群对Cry1Ac敏感性变异小于6倍。采自黄河流域棉区的10个棉铃虫田间种群对Cry1Ac活化毒素的敏感性(LC50:0.063～0.162μg cm-2)略低于采自新疆棉区的2个田间种群(LC50:0.027～0.049μg cm-2).尽管这几年我国主要棉区棉铃虫对Cry1Ac的敏感性没有发生明显变化,但是棉铃虫Bt抗性的检测工作必须加强,开发可靠的抗性基因频率检测技术尤为重要。更多还原

【Abstract】 Cotton bollworm. Helicoverpa armigera (Hubner) (Lepidioptera: Noctuidae) is one of the most important agricultural pests in the old world including China. The serious outbreaks of cotton bollworm in the cotton areas in China caused tremendous economic losses. Since 1980s, the massive use of chemical insecticides resulted in serious resistance problem in cotton bollworm. The resistance of cotton bollworm, especially to pyrethroids was one of the reasons for the outbreaks of cotton bollworm in Northern China during 1990s. Cotton bollworm has been controlled effectively since the Bt cotton was planted in China. But continuous expression of Bt toxin in the whole development time of cotton plant and more than ten-year planting of Bt cotton will impose the cotton bollworm under a strong selection pressure by Bt toxin and increase the risk of resistance of cotton bollorm to Bt cotton.Under selection in the lab, many strains of cotton bollworm developed resistance to Bt toxins. Evolution of Bt resistance by cotton bollworm in the field will affect the longevity of Bt cotton. The studies on the mechanisms of Bt resistance could provide guidance on the management of cotton bollworm. Cadherin is one of the important receptors in Bt action and the mutation of cadherin may cause the resistance of pests to Bt toxin. So far, at least in 3 pests the mutations of cadherin were associated with the resistance to Bt toxin. Particularly, disruption of the cadherin gene (Ha_BtR) by a premature stop codon was associated with a high level of Cry1Ac resistance in H. armigera. In this study, the mutations of cadherin involved in Cry1Ac resistance were researched in both the laboratory-selected GYBT strain and field-collected populations of H. armigera. The study will facilitate understanding of the molecular mechanism of Cry1Ac action in cotton bollworm and prediction of the mechanism of resistance of cotton bollworm to Cry1Ac. The work will also provide technical support on Bt resistance monitoring and resistance management in cotton bollworm.1. Identification and detection of a mutated cadherin allele in the laboratory-selected GYBT strain of H. armigeraThe genomic organization of Ha_BtR was compared from both the susceptible and resistant strains and a deletion was found to be responsible for a truncated cadherin protein in the resistant GYBT strain. Determination of the genomic DNA sequences of Ha_BtR gene showed that the wild type Ha_BtR coding sequence (Genbank No:DQ523166) is comprised of 34 exons. A deletion between Exon 8 and Exon 25 was found to be responsible for a truncated cadherin (Genbank No:DQ523167) in the Cry1Ac-resistant GYBT strain of H. armigera. A DNA-based detection method specific to the r1 allele was developed. This study will facilitate the monitoring of cadherin mutant frequency in field populations of H. armigera.2. Identification and detection of Ha_BtR mutations in field populations of H. armigeraA deletion mutation allele (r1) of a cadherin gene (Ha_BtR) was previously identified as genetically linked with Cry1Ac resistance in a laboratory-selected strain of H.armigera. Using a biphasic screen strategy, we successfully trapped two new cadherin alleles (r2 and r3) associated with Cry1Ac resistance from a field population of H.armigera collected from Anyang, Henan province of China in 2005. The r2 and r3 alleles, respectively, were created by inserting the long terminal repeat of a retrotransposon (designated HaRT1) and the intact HaRT1 retrotransposon at the same position in exon 8 of Ha_BtR, which results in a truncated cadherin containing only two ectodomain repeats in the N terminus of Ha_BtR. This is the first time that the Bt resistance alleles of a target insect of Bt crops have been successfully detected in the open field. This study also demonstrated that bollworm larvae carrying two resistance alleles can complete development on Bt cotton. The cadherin locus should be an important target for intensive DNA-based screening of field populations of H.armigera.Using an F1 screen method,10 candidate and 4 putative candidate single-pair families were selected for Ha_BtR mutation analyses from a field population of H. armigera collected from Langfang, Hebei Province of China in 2009. Through cloning and sequencing of cadherin alleles, at least 5 new mutated alleles of Ha_BtR were found compared with the wild cadherin.117#:an insertion resulting in 30 animo acid insertion at 754aa or premature stop at 751aa; 239#:705bp deletion caused 235 animo acid deletion in the EC11-CYT domain; 234#:alternative splicing caused deletion of the EC2-6 domain; 256#:4 animo acid YTIT deletion; 124#:165bp deletion resulting in deletion of 55 animo acids in the CYT domain. These five mutations associated with Cry1Ac resistance can impair the normal fuction of cadherin. Cadherin alleles with amino acid substitutions were also found to be associated with Cry1Ac resistance. The role of the animo acid substitutions should be investigated in future. In conclusion, the mutation diversity of Ha_BtR will complicate resistance allele frequency monitoring in H. armigera.3. Construction of a pair of near-isogenic strains with and without Ha_BtR r1 allele and its cross resistance patternThe r1 allele of Ha_BtR was introgressed into a susceptible SCD strain by crossing the GYBT strain to the SCD strain, followed by repeated backcrossing to the SCD strain and molecular marker assisted family selection. The introgressed strain (designated as SCD-r1, carrying homozygous r1 allele) obtained 438-fold resistance to Cry1Ac,> 41-fold resistance to Cry1Aa and 31-fold resistance Cry1Ab compared with the SCD strain; however, there was no significant difference in susceptibility to Cry2Aa between the integrated and parent strains. It confirms that the loss of function mutation of Ha_BtR alone can confer medium to high levels of resistance to the three Cry1A toxins in H. armigera. Reciprocal crosses between the SCD and SCD-r1 strains showed that resistance to CrylAc in the SCD-r1 strain was completely recessive.Modified toxin Cry1AcMod, CrylAbMod and the protoxin CrylAcPro and Cry1AbPro countered the resistance in the resistant SCD-r1 strain. Resistance to Cry1AcMod, Cry1AbMod, Cry1AcPro and Cry1AbPro was significantly reduced when compared with resistance to the activated Cry1Ac and Cry1Ab respectively. These modified toxins which can counter cadherin-based resistance may become the candidate genes engineering into the transgenic cotton.4. Susceptibility monitoring of field populations of H. armigera to Cry1AcUsing the toxin overlay method, susceptibilities of H. armigera populations from main cotton areas of China were monitored during 2006-2008. Limited variation (less than 6-fold) in Cry1Ac susceptibility was found in the 12 field populations sampled during 2006-2008. Ten populations (LC50:0.063～0.162μg cm-2) collected from Yellow River cotton area showed less susceptibility than that of two populations (LC50:0.027～0.049μg cm-52) collected from Xinjiang cotton area. Although there are no signifianct changes in Cry1Ac susceptibility in the field populations of H. armigera colleted in this study, we should keep an eye on resistance evolution of H. armigera to Bt cotton in China. Development of effective techniques for resistance gene frequency detection should be a priority for Bt resistance research in H. armigera.更多还原